synthesis and fabrication of surface-active microparticles ... · synthesis and fabrication of...
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Communication
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Synthesis and fabrication of surface-active microparticles using membrane
emulsion technique and rapid conjugation of model protein via strain-promoted
azide-alkyne click chemistry in physiological conditions
Grace Walden a, Xin Liao a, Graham Riley b, Simon Donell c, Michael J Raxworthy d and Aram Saeed a*
a School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, United Kingdom. b
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom. c Norwich Medical School, University of East Anglia , Norwich, NR4 7TJ, United Kingdom. d Neotherix Limited, York, YO1 7PR, United Kingdom.
Abstract: The rapid surface immobilisation of protein on monodispersed polyester microcarriers is reported. A model protein,
functionalised with a dibenzocyclooctyne core, immobilises on the surface of azide-terminal polycaprolactone microcarriers within 10
minutes compared to 12 hours for other conjugation techniques and is conducted in physiological conditions and in the absence of
coupling reagents.
In the field of tissue engineering and regenerative medicine,
there is more demand for biomaterials that are able to provide the
necessary biological cues needed to establish a natural healing
environment.1,2 Therefore the design of simple and effective
delivery systems for bioactive molecules, including proteins and
cells, is becoming increasingly important.3 A delivery system
that is able to exert spatiotemporal control over the release and
presentation of biomolecules, localised to the site of action, could
minimise side effects, increase therapeutic effectiveness and
enhance cellular retention.4,5 An effective polymeric delivery
system for proteins needs to be biodegradable at a rate
comparable to the re-establishment of new tissue, and material
properties.6 In addition, it needs to demonstrate little to no
cytotoxicity and avoid generating an inflammatory response.27
Polymers used for delivery systems need to contain biomimetic
adhesion sites for the facile conjugation of bioactive agents.8
Currently, research towards the synthesis of these types of
delivery systems has been met with the challenge of choosing
between natural polymers abundant with biologically active
interaction sites or mechanically superior synthetic polymers
lacking in useful functional groups.9
Synthetic polymers have advantageous chemical properties, such
as good porosity, tuneable degradation rates and increased
mechanical strength.10 These polymers, however, suffer from
poor surface adhesion and an inability to easily conjugate
biomolecules such as proteins or peptides that can interact with
cellular components.9 For decades, polyester polymers such
poly(lactic-co-glycolic acid) (PLGA), Poly(caprolactone) (PCL)
and poly(lactic acid) (PLA) have been the preferred materials for
delivery systems.19,11 However, these polymers are inherently
difficult to work with. They lack functional groups needed to
allow for easy conjugation to, or control the adhesion of,
bioactive agents.12,13 This, in turn, has limited their application.12
Post-functionalisation of these polymers, in which functional
groups are substituted or modified using additional reagents, has
been attempted to combat this problem.14 One of the most
common techniques employed to functionalise these polymers is
aminolysis.15 This requires physical adsorption on the surface
and the use of carbodiimide coupling reagents.1 However, as
these techniques often require multi-step reactions and laborious
purification processes, these delivery systems have seen little
success in clinical translation.16 Recently, a new approach has
been developed in which functional ε-caprolactone monomers
can be used to synthesise polycaprolactone with a variety of
functional groups.13 This is achieved by utilising the substitution
of halogen groups or functionalisation of the carbonyl backbone
of PCL by bromination, epoxidation or hydroxylation.13 To meet
these challenges, we introduce a facile way to control the
functional groups present on the polymer PCL by changing the
initiator used during ring opening polymerisation (ROP). This
functionalised polymer is then able to carry out the click
chemistry reaction for the efficient conjugation of protein.
A heterobifunctional initiator containing a hydroxyl group at one
end, and an azide group at the other can be used for the
polymerisation of ɛ-caprolactone in bulk, with a catalytic amount
of tin(II) 2-ethylhexanoate at 120oC and under an atmosphere of
nitrogen. This resulted in PCL functionalised with a terminal
azide (Figure 1, A).
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For the ring opening polymerisation, 2[2-[2-
azidoethoxy)ethoxy]ethanol was used as the heterobifunctional
initiator, containing a short diethylene glycol spacer to enhance
its hydrophilicity. 1HNMR diffusion ordered spectroscopy
(DOSY) showed that the terminal azide of PCL could readily
bind to a dibenzocyclooctyne (DBCO) unit within 30 minutes
(see supporting information, S5). The structure and molecular
weight of the purified polymer was confirmed by nuclear
magnetic resonance (NMR) and gel permeation chromatography
(GPC). The 1HNMR showed signature peaks indicative of a
polycaprolactone backbone. The number average molecular
weight (Mn) of the polymer was confirmed by GPC from
samples measured in triplicate and averaged as 19502, with
typical dispersity index (PDI) for ROP of 1.1, indicative of a
controlled polymerisation reaction and a uniform polymer, in
agreement with published work.17 Fourier transform infrared
spectroscopy (FTIR) analysis of the purified PCL-azide polymer
showed a peak present at 2101 cm-1, confirming the successful
inclusion of the azide initiator into the backbone of the polymer
(data not shown).
Next, the PCL-azide polymer was used for the fabrication of
monodispersed microparticles using an oil in water membrane
emulsification technique (Figure 1, B). Briefly, the membrane
emulsification kit contains a glass cylinder that sits atop the
injection chamber, which holds a metal membrane with uniform
pores of a specified size throughout. PCL-azide polymer
dissolved in dichloromethane (DCM) was used as the oil phase
and injected via an automated syringe pump through the pores of
the membrane into the aqueous phase containing 1% polyvinyl
alcohol (PVA) as a surfactant. The PCL-azide droplets form on
the surface of the membrane and are removed by a stirrer paddle
Figure 1: Reaction scheme showing the synthesis of functionalised microparticles with protein immobilised on the surface. Step A shows the synthesis of a functionalised PCL-azide
polymer. This is then used as the oil phase, dissolved in organic solvent, to produce microparticles via oil and water emulsion (step B). Step C shows the conjugation of protein to
DBCO-peg4-maleimide which attaches to free thiols present within the protein. This reaction product is then used to conjugate to microparticles as shown in step D utilizing strain
promoted azide alkyne cycloaddition.
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with adjustable rotation speed. PCL-azide droplets gradually
solidify, by solvent evaporation, resulting in microparticles that
can be washed to remove all PVA and freeze-dried for further
use. It is possible to tightly control particle size and
monodispersity by altering the process parameters. The effect of
increasing rotation speed and polymer concentration on particle
morphology and size distribution was investigated. Polymer
concentrations of 5, 10, 20, 30 and 40w/v% PCL-azide in DCM
were used and the rotation speed kept constant at 1140 RPM. It
was found that as the polymer concentration increased so did the
average particle size, resulting in particles of 24±0.21 µm and
34±0.39 μm in diameter for 5 and 40w/v% PCL-azide
respectively (Figure 2).The span of particles represent the size
distribution, and can be calculated using (D90-D10)/D50) where
D represents the diameter which is equal to or greater than that
% of particles in the sample.18,10 A size distribution of less than
1 indicates a monodisperse population of particles.
At all PCL-azide concentrations the size distribution of particles
was low at less than 0.5 indicating a highly monodisperse
population (see supporting information, S2). The increase in
particle size can be attributed to the increased viscosity, due to
increased concentration of the polymer, which results in larger
droplets forming on the surface of the membrane before
detachment by the rotating stirrer paddle.19 Rotation speeds of
400, 590, 770, 950, 1140 and 1500 RPM were assessed, whilst
polymer concentration remained constant at 10 w/v%. It was
found that as rotation speed increased the average particle size
decreased, resulting in particles with a diameter of 71±2.18 μm
and 22±0.24 μm at rotation speeds of 400 and 1500 respectively.
At the lowest RPM (400) it was found that, the dispersity of the
microparticles was poorest, with a span of 1.2, suggesting a less
monodispersed population, but this improved when the stir speed
was increased (see supporting information, S2). A decrease in
particle size with increased rotation speed is due to the rapid
collection of particle droplets from the surface of the
membrane.19 It was shown that highly monodispersed particles
with a uniform, spherical morphology could be produced using
membrane emulsification. Precise control over the resulting
Figure 2: PCL-azide dissolved in DCM was injected into 1%w/v PVA solution via syringe injection at a controlled rate. Particles were produced using either increasing stir speeds
(Rows A-C) or increasing polymer concentrations (Rows D-F). Images show fresh particles (Rows A and D), solidified particles (Rows B and E) and freeze-dried particles visualised
using Scanning electron microscopy (Rows C and F). All optical microscopy images taken at 10x objective. SEM shown at x190 magnification. All scale bars represent 100µm.
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particle size was possible. The resulting particles were assessed
for the presence of azide using IR and were all found to contain
the indicative azide peak at 2100 cm-1, showing no degradation
during the production process (data not shown).
To demonstrate the application of PCL-azide particles for the
delivery of bioactive factors, we functionalised the model protein
Human Serum Albumin (HSA) with a click chemistry unit,
containing an internal alkyne, which can readily react with the
azide functionality of PCL particles (Figure 1, D). With one-step
modification in mind, we opted to use a copper-free click
chemistry reagent, which is commercially available.
Dibenzylcyclooctyne-peg4-maleimide (DBCO-mal) is readily
available to react with the free thiol present in the cysteine at
residue 34 of HSA (Figure 1, C).20 The resulting conjugate was
analysed by HPLC (see supporting information, S3) and liquid
chromatography mass spectrometry (LC-MS) (see supporting
information, S4). The native unconjugated protein was identified
by a sharp peak at 66559Da MW by LC-MS as expected.21 The
conjugation reaction was successful with 73% relative
abundance of the protein labelled, as shown by the additional
peaks increasing in MW by 680Da corresponding to the addition
of DBCO-mal (see supporting information, S4).
By labelling HSA with a fluorescent tag, we were successfully
able to monitor the speed and efficiency of the click reaction of
DBCO functionalised protein onto the surface of PCL-azide
microparticles. HSA was tagged with fluorescent isothiocyanate
and DBCO-mal to produce a fluorescently tagged protein that
could be used in the click reaction. All unbound reactants were
removed using a PD10 desalting column and the fluorescent
conjugates were mixed with PCL-azide Microparticles at a ratio
of 1:1 mole equivalents (in respect to the polymer) for 10
minutes, 1 hour, 2 hour, and 12 hour time points. After reaction,
the particles were washed with distilled water and fluorescent
images were taken (Figure 3). It was found that the
immobilisation of protein onto the surface of the PCL-azide
microparticles occurred within 10 minutes compared to 12 hours
for non-functionalised PCL controls. Microparticles displayed
fluorescence and therefore the presence of HSA protein at all
time points (Figure 3).
Azide-deficient PCL, which lacks the reactive groups needed for
the attachment of the DBCO linker, was used as a control.
Fluorescence was only seen on these particles after 12 hours; by
this point physical absorption of water onto the surface of the
particles could be responsible for the fluorescence seen, as
opposed to a conjugation reaction occurring.
To summarise, here we describe a readily adaptable template for
the immobilisation of biological factors that can be applied to a
large variety of widely available polyester polymers. We have
demonstrated the facile production of surface-clickable
microcarriers with the simple synthesis of commercially
available polymers, functionalised with useful terminal groups.
We have demonstrated the one step copper-free click chemistry
reaction can be carried out with high efficiency in 10 minutes in
physiological conditions. It can be used for the immobilisation
of proteins and other bioactive factors to the surface of a
microcarrier delivery system. The DBCO reagent is
commercially available with a variety of reactive functional
groups that can be used to conjugate to amines, carbonyls, and
thiols. This means that the techniques discussed here can easily
be adapted and adjusted to allow conjugation to a whole range of
compounds. The combination of surface active microcarriers
with efficient and specific DBCO reagents offers a new way of
loading and delivering bioactive agents, with potential
applications in both drug delivery and tissue engineering.
CONFLICTS OF INTEREST
There are no conflicts to declare
ACKNOWLEDGEMENTS
The authors are grateful for the funding provided by the
EPSRC ICASE award (grant EP/L505729/I).
SUPPORTING INFORMATION
Detailed experimental procedures and additional figures;
synthesis of the PCL-Azide polymer; Strain promoted Azide-
Alkyne Cycloaddition; Microparticle production; synthesis of
DBCO-peg4-maleimide-HSA conjugates; synthesis of HSA
immobilized PCL-N3 microparticles; HPLC analysis of HSA and
DBCO-peg4-maleimide; LCMS analysis of the HSA
conjugation with DBCO-peg4-maleimide; DOSY-NMR spectra
of DBCO-acid and corresponding reaction of DBCO-acid with
PCL-N3 and PCL.
AUTHOR INFORMATION Corresponding author
Figure 3: PCL-azide and PCL microparticles reacted with FITC labelled Human Serum
Albumin at 1:1 molar ratio for increasing amounts of time. PCL-azide contains azide
functional groups, which can react with the internal alkyne present on the DBCO linker
tethered to the Human Serum Albumin via free cysteine. This copper- free click chemistry
reaction occurs quickly and microparticles are fluorescent after just 10 minutes.
Commercial PCL lacks the azide functional group, but fluorescence occurs if the reaction
is carried out for a sufficient length of time to allow physical adsorption. All Images taken
at x20 magnification. All scale bars represent 100µm.
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Notes
The Authors declare no competing financial interest
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